The bacterium Bacillus thuringiensis (Bt) contains genes encoding insecticidal proteins. Bt proteins are toxic when ingested by susceptible insect and insect larvae. Bt proteins are used commercially in pesticide formulations, and plants transformed with Bt genes provide transgenic crop plants whose cells produce the insecticidal protein. The Bt gene codes for a protein toxin that attacks the insect midgut, stops feeding and eventually kills susceptible insects. Gill et al., Annu. Rev. Entomol. 37:615 (1992); Fischhoff, In Biotechnology and Integrated Pest Management, Ed. GJ Persley, pp. 214-227, CAB International, Cambridge, UK.
Several hundred strains of Bacillus thuringiensis exist, with considerable specificity toward various groups of insects such as the lepidoptera (butterflies and moths), coleoptera (beetles) and/or diptera (mosquitoes), as well as toward nematodes. Coevolution of insects and Bt has resulted in specificity of the interaction between Bt toxin and the membranes of insect gut cells. The Bt toxin of a particular B. thuringiensis strain may bind to the gut of lepidopteran larvae, or only some species of lepidopteran larvae, but not to others. Binding of the protein to the membrane is required for its toxic effects. Thus the Bt toxins have a high specificity for a small number of pest species, while having no significant activity against beneficial insects, wildlife or humans. Lambert and Peferoen, BioScience, 42:112 (1992); Gill et al., Annu. Rev. Entomol. 37:615 (1992); Meadows, In: Bacillus thuringiensis, An Environmental Biopesticide: Theory and Practice, Entwistle et al., Eds., pp. 193-200 (1993).
Formulations of Bt toxin for use as insecticides are known in the art. See, e.g., U.S. Pat. No. 5,747,450; U.S. Pat. No. 5,250,515; U.S. Pat. No. 5,024,837; U.S. Pat. No. 4,797,276; and U.S. Pat. No. 4,713,241.
Plants transformed to carry the Bt gene and express insecticidal proteins are known in the art, and include potato, cotton, tomato, corn, tobacco, lettuce and canola. Krimsky and Wrubel, Agricultural Biotechnology: An Environmental Outlook, Tufts University, Department of Urban and Environmental Policy, p. 29 (1993). See also U.S. Pat. No. 5,608,142; U.S. Pat. No. 5,495,071; U.S. Pat. No. 5,349,124; and U.S. Pat. No. 5,254,799. The use of such genetically engineered plants is expected to reduce the use of broad spectrum insecticides. Gasser and Fraley, Science 244:1293 (1989).
The use of pesticides results in the selection of individuals resistant to the pesticide, and can lead to the development of pesticide-resistant populations. Resistance to chemical insecticides such as organophosphates, carbamates, spinosyns and pyrethroids are known. Laboratory and field evidence documents that many pests are capable of evolving high levels of resistance to a number of commonly used Bt toxins. Tabashnik, Annu. Rev. Entomol. 39:47 (1994); Tabashnik, J. Econ. Entomol. 83:1671 (1990); Bauer, Fla. Ent. 78:414 (1995); Gould, Proc. Natl. Acad. Sci. USA 94:3519 (1997). Resistance may evolve whether the Bt is applied to plants or the plants are genetically engineered to express Bt. The development of resistance to Bt toxin-expressing crops may also result in resistance to commercial formulations of fermented strains of Bt, such as DIPEL.RTM. (Abbott Laboratories).
A further concern in the use of plants genetically engineered to express Bt toxins is the difficulty of distinguishing between different pest species that will and will not be controlled by Bt. The presence of a pest in the field that is resistant to Bt indicates the need for supplemental pesticide treatments, whereas no additional treatment is needed if pests are susceptible to Bt. In the case of cotton, transgenic Bt cultivars are exceptionally toxic to most strains of the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) (Jenkins et al., J. Econ. Entomol. 86:181 (1993)), but are less toxic to the bollworm Helicoverpa zea (Boddie)(Lepidoptera:Noctuidae) (Lambert et al., In: Proceedings Beltwide Cotton Conference, pp. 931-935, National Cotton Council, Memphis Tenn. (1996)). H. zea and H. virescens are found in the same geographic areas, and in years when H. zea populations are high, larva that are not killed by ingestion of Bt can cause significant damage to cotton. The eggs and young larvae of H. zea and H. virescens are indistinguishable by simple observation in the field (although adults are readily distinguished visually). Without a test to distinguish among susceptible and resistant species, farmers finding lepidopteran eggs or neonates on cotton cannot rely on Bt cotton for control of lepidopteran pests.
Rapid, reliable methods to distinguish Bt-susceptible from Bt-resistant species, and to detect the development of Bt resistance in populations of insects, are desirable. The methods of the present invention provide a bioassay capable of distinguishing between H. virescens and H. zea. The present methods can also detect insect resistance to Bt within a species.